Several types of ribozymes have been identified in living organisms. One of the first ribozymes to show catalytic turnover was the RNA moiety of ribonuclease P. Ribonuclease P (RNase P) cleaves precursor tRNAs (pre-tRNAs) at their 5' ends to give the mature 5'-termini of tRNAs. In Escherichia coli and Bacillus subtilis, the RNase P holoenzyme is composed of one basic protein subunit of approximate M.sub.r 14,000 (119 amino acids) and one single stranded RNA molecule of 377 and 401 nucleotides, respectively [Baer, 1990; Altman 1987; Waugh, 1989; Pace, 1990; Nichols, 1988]. Another early ribozyme to show cleavage was the L-19 intervening sequence (IVS) from tetrahymena. The 413 nucleotide intervening sequence (IVS) in the nuclear rRNA precursor from Tetrahymena thermophila can be excised and the two exons ligated in the complete absence of any protein [Kruger, 1982; Cech, 1981]. Unique to this class of self-splicing reaction is the requirement of a guanosine or 5' guanosine nucleotide cofactor. The hammerhead, which in nature undergoes a self-cleavage reaction, constitutes a third class of ribozymes. A number of plant pathogenic RNAs [Symons, 1989; Symons, 1990; Bruening, 1989; Bruening 1990], one animal viral RNA [Taylor, 1990] and a transcript from satellite II of DNA of the newt [Epstein, 1987; Epstein 1989] and from a Neurosoora DNA plasmid [Saville, 1990] undergo a site specific self-cleavage reaction in vitro to produce cleavage fragments with a 2',3'-cyclic phosphate and a 5'-hydroxyl group. This reaction is unlike RNase P RNA cleavage of pre-tRNAs, where the internucleotide bond undergoes a phosphoryl transfer reaction in the presence of Mg.sup.++ or other divalent cations. Metal cations may be essential to RNA catalysis [Pyle, 1993]. Other reactions documented to date show that ribozymes can catalyze the cleavage of DNA [Robertson, 1990; Herschlag 1990], the replication of RNA strands [Green, 1992], the opening of 2'-3'-cyclic phosphate rings [Pan, 1992], as well as react with phosphate monoesters [Zaug, 1986] and carbon centers [Noller, 1992; Piccirilli, 1992]. Finally, ribozymes with new kinds of catalytic reactivity are being created through techniques of in vitro selection and evolution [Breaker and Joyce, 1994; Szostak, 1992].
The ability to design a ribozyme to specifically target and cleave any designated RNA sequence ha, led to much interest in the potential application of hammerhead ribozymes in transgenic plants and in animal health as gene therapy agents or drugs. To improve the ability to treat a disease or target a specific nucleic acid, it is desirable to optimize the ribozyme to achieve the maximum cleavage activity. While much success has been achieved in vitro in targeting and cleaving a number of designated RNA sequences (Saxena and Ackerman, 1990; Lamb and Hay, 1990; Evans, et al., 1992; Mazzolini, et al., 1992; Homann, et al., 1993), there are fewer whole cell examples.
Previous reports have demonstrated that high levels of ribozyme expression are required to achieve reduced accumulation of target sequence in vivo [Cameron and Jennings, 1989; Cotten and Birnsteil, 1989; Sioud and Drilca, 1991; L'Huillier, et al., 1992; Perriman et al., 1993]. Another article suggests a necessity for the target and ribozyme to be sequestered in the same cellular compartment [Sullenger and Cech, 1993]. These reports demonstrate that hammerhead ribozymes are clearly capable of specific cleavage of a designated target RNA within a biological system.